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NanoDLSay: Nanoparticle-Enabled Dynamic Light Scattering Assay for Chemical and Biological Detection and Analysis Copyright Nano Discovery Inc. 2012 www.nanodiscoveryinc.com Tel: 407-770-8954 Email: sales@nanodiscoveryinc.comwww.nanodiscoveryinc.comsales@nanodiscoveryinc.com July 2012 One New Technology, Discover a New World

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Part I. General introduction Part II. NanoDLSay for protein research Part III. Comparison with other analytical techniques Part IV. NDS1200 the instrument for NanoDLSay

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Part I. General Introduction o The principle of NanoDLSay o How to conduct NanoDLSay o Applications and examples o Analytical performance and advantages

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What is NanoDLSay: Detect the target analytes by monitoring the size change of nanoparticles upon binding with the target analyte AuNP immunoprobe D 120 nm AuNP immunoprobe bound with a small protein monomer D 130-160 nm AuNP immunoprobe bound with a large protein complex D > 130-160 nm Y Y Y Y Y Y Y Y Y Y Y Y Y Y AuNPs bound with metal ion targets through metal-chelating ligands AuNPs bound with small chemical targets through coordinative ligand interactions Unmodified AuNP D = 100 nm D >> 100 nm 2+ Y Y Y Y Y Y Y General assay format: AuNP clusters formed from binding with target analytes D >> 100-200 nm Gold nanoparticle (AuNP)

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Two assay formats Y Y Y Y Y Y Y I. Individual particle size increase Suitable for large analytes such as proteins, complexes and viruses Suitable for kinetic binding studies II. Nanoparticle cluster formation Suitable for any analytes, especially for chemicals and ions Provide best sensitivity Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

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What is dynamic light scattering (DLS): Measure particle size in nanometer size range Scattered light intensity fluctuation Correlation function Laser beam Scattering light NDS1200 Instrument

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Why Gold Nanoparticles (AuNPs)? Exceptionally intense light scattering property 10 5 times stronger than a fluorescent dye molecule; 100s times stronger than polystyrene (PS) latex particles Detection limit of DLS for AuNPs can easily reach fM to aM range As an optical probe, AuNPs easily stands out from sample matrix AuNPs Serum A AuNPs PS particle B C Gold nanorods Dark field optical images of AuNPs mixed with human serum (A) and PS particles (B). (C) A dark field optical image of gold nanorods (AuNR)

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How to conduct NanoDLSay? Step 1. Prepare the AuNP probe Step 2. Mix the AuNP probe with the sample solution Step 3. Incubate the assay solution Step 4. Measure the particle size of the assay solution A typical assay condition: 1.Mix 40 L AuNP probe with 2 L sample 2.Incubate 5-15 min at room temperature 3.Analyze the particle size to obtain results Read-out: average particle size (nm) Dose-response curve Target concentration Average particle size (nm) Unknown sample Standard curve

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AuNP Bioconjugate Preparation 1. Direct adsorption method Citrate AuNPs (100 nm) (Ted Pella Inc.) (1 mL AuNP + 5-10 g antibody) Blocking reagent: Bovine serum albumin (BSA) (2.5 mg/mL) 1.Centrifuge 2.Re-dispersion ~15 min ~30 min 2. Covalent conjugation method -NH 2 or -COOH NHS/EDC activation 1.Centrifuge 2.Re-dispersion Functional ligand-coated AuNPs Easy to use but lower stability; primary choice More complicated but higher stability

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Applications of NanoDLSay Proteins DNAs RNAs Viruses Small chemicals Toxic metal ions

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Examples Liu X, et al. A One-step homogeneous immunoassay for cancer biomarker detection using gold nanoparticle probes coupled with dynamic light scattering. J. Am. Chem. Soc. 2008; 130:2780-2782. Chun C, et al. A facile and sensitive immunoassay for the detection of alpha- fetoprotein using gold-coated magnetic nanoparticle clusters and dynamic light scattering. Chem. Comm. 2011, 47, 11047-11049. Driskell JD, et al. One-step assay for detecting influenza virus using dynamic light scattering and gold nanoparticles. Analyst 2011; 136:3083-3090. Kalluri JR, et al. Use of gold nanoparticles in a simple colorimetric and ultrasensitive dynamic light scattering assay: selective detection of arsenic in groundwater. Angew. Chem. Int. Ed. 2009; 48:9668-9671. Gao D, et al. An ultrasensitive method for the detection of gene fragment from transgenics using label-free gold nanoparticle probe and dynamic light scattering. Anal. Chim Acta 2011; 696:1-5. Wang X, et al. Detection of hepatitis B surface antigen by target-induced aggregation monitored by dynamic light scattering. Anal. Biochem. 2012, online. For a more complete list, visit: www.nanodiscoveryinc.comwww.nanodiscoveryinc.com

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Analytical Performance AnalytesSensitivityDynamic Range ProteinsHigh pg/mL to low ng/mL range2-3 orders of magnitude DNAs30 fM (5 orders of magnitude more sensitive than SPR and fluorescence techniques) > 5 orders of magnitude Viruses< 100 TCID 50 /mL (1-2 orders of magnitude more sensitive than commercial diagnostic kits) 2-3 orders of magnitude Toxic metal ionsArsenics: 10 ppt (WHO acceptable limit: 10 ppb) Lead: 100 ppt (2 orders of magnitude below the EPA standard limit) 2-3 orders of magnitude Small molecules7 nM (5 orders of magnitude more sensitive than the colorimetric method) > 4 orders of magnitude Explosive chemicals100 pM2-3 orders of magnitude Notes: (1) ng-nanogram; fg-femtogram; fM-femtomolar; pM-picomolar; nM-nanomolar; ppb-parts per billion; ppt-parts per trillion; TCID 50 - 50% tissue culture infective dose. (2) All data were taken from published papers. Refer to the list of publications for more information. (3) WHO: World Health Organization; EPA: Environmental Protection Agency.

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Ref: Gao D, Sheng Z, Han H. An ultrasensitive method for the detection of gene fragment from transgenics using label-free gold nanoparticle probe and dynamic light scattering. Anal. Chim Acta 2011; 696:1-5. LabelMethodDetection limit AuNPColorimetric1 10 -8 mol/L Au chipSurface plasmon resonance1 10 -9 mol/L Au/polyaniline nantube Electrochemical impedance spectroscopy 3 10 -13 mol/L Quantum dotsAnodic stripping voltammetry5 10 -11 mol/L ZnS and CdSe quantum dots Fluorescence2 10 -9 mol/L NanoDLSayDynamic light scattering3 10 -14 mol/L Comparison of NanoDLSay with other methods for DNA detection Analytical Performance

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The ultrahigh sensitivity of NanoDLSay AuNP monomer versus clusters Size100 nm~300 nm Scattered light intensity ratio1~1000 Number (molar) ratio99.9% (10 pM)0.1% (10 fM) Net scattered light intensity11 Intensity-averaged particle size * 100 nm + * 300 nm = 200 nm : Calculated according to Mie scattering theory From the above illustration, it can be seen that with a trace amount of AuNP cluster formation due to target analyte binding, the intensity-averaged particle size increases substantially - The origin of high sensitivity of NanoDLSay

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Advantages of NanoDLSay o Requires a small volume of sample (1-5 L) o Obtain results in several minutes o Single-step assay procedure o Extremely simple and easy to use o High to ultra-high sensitivity o Excellent reproducibility o Extremely low cost of consumables

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Part II. NanoDLSay for Protein Research o Introduction o Protein detection and concentration analysis o Kinetic study of protein-protein interaction o Label-free protein complex detection and binding partner analysis o Label-free protein oligomer/aggregate detection and analysis

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Introduction: Understand the problems of traditional immunoassay X B Traditional immunoassay likely fails to detect proteins in complexes A Traditional immunoassay assumes proteins exist alone antibody Individual protein monomer Protein complex Protein aggregates A protein does not stay alone in biological systems

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Average particle size increase (nm) Incubation time (min) = 2D of analyte 0 min 30 min 1 2 3 Kinetic binding study: monitor the particle size change continuously during the assay Determine the size of the target analyte at a saturated binding level Determine if a target protein is a monomer, complex, or aggregates Label-free detection: no need to label the target proteins Detection of protein complexes and aggregates from real biological samples NanoDLSay : Detect target proteins in all forms Unique capabilities

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Dose-response curve Target concentration Average particle size (nm) Unknown sample Standard curve 1. Protein detection and concentration analysis Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Single-probe assay Two-probe assay or single polyclonal antibody probe assay (higher sensitivity) o Standard curve is established using standard solutions o Relative quantitation can be done by directly comparing the average particle size Y Y Y Y Y Y Y

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2. Kinetic study of protein-protein interaction Target A Target B Procedure: 1.Immobilize one target A protein to the AuNP 2.Mix the target A-modified AuNP with target B protein 3.Monitor the AuNP size change 4.Binding affinity may be estimated using Langmuir adsorption model Average particle size (nm) Incubation time (min) 0 30 Non-binding proteins Alternative:

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3. Label-free protein complex detection and binding partner analysis Average particle size increase (nm) Incubation time (min) Step 2: Binding partner screening using antibody Step 1: Catch the target Particle size change upon antibody addition c ~ 2D Binding partners Not binding partners Step 1. Determine if a target protein exists as a complex (The final net increase of the AuNP size tells how big the target protein is) Step 2. Analyze the binding partners to the target protein

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4. Label-free protein oligomer/aggregate detection and analysis Average particle size increase (nm) Incubation time (min) = 2D of analyte 0 min 30 min protein monomer oligomers, aggregates Protein oligomer/aggregates cause AuNP probe cluster formation Specific detection of target protein oligomer/aggregates in real samples

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References Protein-protein or other biomolecular interactions Jans H, et al. Dynamic light scattering as a powerful tool for gold nanoparticle bioconjugation and biomolecular binding study. Anal. Chem. 2009; 81: 9425-9432. Austin L, et al. An immunoassay for monoclonal antibody isotyping and quality analysis using gold nanoparticles and dynamic light scattering. American Biotechnology Laboratory 2010; 28: 8, 10-12. Snchez-Pomales G, et al. A lectin-based gold nanoparticle assay for proving glycosylation of glycoproteins. Biotechnology Bioengineering 2012, published online. Wang, X.; Ramstrm, O.; Yan, M. Dynamic light scattering as an efficient tool to study glyconanoparticle-lectin interactions. Analyst 2011, 136, 4174-4178. Label-free protein complex detection and binding partner analysis Jaganathan S, et al. A functional nuclear epidermal growth factor receptor, Src and Stat3 heteromeric complex in pancreatic cancer cells. PLoS One 2011, 6(5):e19605. Label-free protein oligomer/aggregate detection Bogdanovic J, et al. A label-free nanoparticle aggregation assay for protein complex/aggregate detection and analysis. Anal. Biochem. 2010; 45:96-102. Huo Q. Protein complexes/aggregates as potential cancer biomarkers revealed by a nanoparticle aggregation assay. Colloids Surfaces B 2010; 78:259-265.

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Part III. Comparison of NanoDLSay with other analytical techniques o ELISA (enzyme-linked immunoabsorbent assay) o Surface plasmon resonance o Co-immunoprecipitation/immunoblotting o Size exclusion chromatography o Analytical Ultracentrifugation o Colorimetric assay using AuNP probes

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1. NanoDLSay versus ELISA Sandwich ELISA NanoDLSay o Likely fail to detect complexed proteins o Results obtained in 2-3 hours o Multiple steps extensive labor o Relatively large sample volume (10-100s L) o Detect target protein in all forms o Reveal more accurate biological information o Reveal protein complex state o Results obtained in several minutes o Single step process o Samll sample volume (1-5 L)

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2. NanoDLSay versus Surface Plasmon Resonance (SPR) o Label-free technique o Optical substrate: gold nanoparticle o Read-out: AuNP size change o Homogeneous solution assay o Low cost of consumables o Reveal the size information of the target analyte, distinguish protein complexes and oligomers/complexes from monomers o Label-free technique o Optical substrate: gold thin film o Read-out: refractive index change o Heterogeneous chip assay o High cost of consumables o Does not reveal the size information of the target analyte, does not tell whether a protein is a monomer, complex or oligomer NanoDLSay SPR

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3. Comparison of NanoDLSay with co-immunoprecipitation (Co-IP) followed by immunoblotting for protein complex analysis

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Non-specific interactions A problem in Co-IP: o Significant non-specific interactions caused by the separation process o The concentration of the particle probes and proteins is artificially increased during centrifugation, increasing non-specific interactions This problem does not exist in NanoDLSay : o The AuNP probe concentration is relatively low, reducing non-specific interactions o No centrifugation separation is involved

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4. NanoDLSay versus size exclusion chromatography (SEC) and analytical ultra-centrifugation (AU) for protein complex and oligomer/aggregate detection and analysis SEC and AU: o For pure protein solution study only o SEC underestimates complex or oligomer/aggregate formation (eluent dilution disrupts existing complexes/oligomers) o AU overestimates complex or oligomer/aggregate formation (centrifugation artificially increases protein complexes/oligomers) NanoDLSay : o Detect protein complexes, oligomers/aggregates from real samples o Fast screening test for protein complex/oligomer/aggregates o Not suitable for absolute quantitative analysis of various oligomers

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5. Comparison of NanoDLSay with colorimetric assay Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Color change Wavelength (nm) Absorbance (A.U.) 400 800 Before assay After assay 600 500 700 Colorimetric assay o Easy to perform o With or without instrument o Low sensitivity o Does not reveal molecular size information o Not suitable for colored samples (e.g. blood) Target analyte binding-induced AuNP cluster formation causes SPR band shift of AuNPs to longer wavelength - Color change

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Part IV. NDS1200: A new dynamic light scattering instrument designed for performing NanoDLSay Product & Services Automatic measurement of 12 samples Automatic kinetic study of 12 samples Fast analysis time: 10-20s per sample 40 L assay solution is used for the measurement Low-cost, disposable min-glass tubes with caps are used as sample containers. No cross-contamination between samples High throughput analysis capability: 120- 180 samples/hour The hardware is maintenance-free No special housing environment is required for the instrument Extremely easy-to-use software

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Product & Services NanoDLSay software: A software designed for convenient, flexible and high throughput analysis

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Notes o Patent application pending on NanoDLSay technology and NDS1200 system: PCT/US09/030087 and PCT/US11/21002 o Nano Discovery Inc. has the exclusive license in the world to practice and commercialize NanoDLSay technology Please Contact Us to Request a Quote: NDS1200 Dynamic light scattering instrument for conducting NanoDLSay Assay kit including disposable sample cells and other consumables 3251 Progress Drive Suite A1 Orlando, FL 32826 Phone: 407-770-8954 Email: sales@nanodiscoveryinc.com NDS-Kit1000 Order Information Or visit online: www. nanodiscoveryinc.com Product and Order Information